Overview
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The surface of the cornea abuts directly up against the surrounding conjunctiva. It is not clear how the boundary between these two distinctly different tissues is maintained. This is an important question since common diseases affecting the eye often destroy the cornea-conjunctiva barrier. In such cases, the conjunctiva spreads across the surface of the eye, thus impairing vision. The present hypothesis is that cells derived from the surface of the cornea and conjunctiva respectively will display evidence of boundary-forming function in vitro, and that this property is related to production of molecules which belong to the Eph/ephrin family of proteins.
Aims, objects and expected outcomes
1. To examine the behaviour of corneal epithelial cells and conjunctival epithelial cells when cultured together in vitro.
Our hypothesis is that the two cell types will preferentially avoid contact with each other owing to the expression of barrier-forming cues such as those provided by Eph/ephrin interactions in other cell types. Importantly, the hypothesis is not dependent upon Eph/ephrin molecules being responsible for this behaviour.
Rationale
Under normal circumstances, the conjunctiva is not able to migrate across the surface of the cornea. Current research, including that by the Ocular Cell Biology Program at QUT1, demonstrates that this patterning is due at least in part to the presence of epithelial progenitor cells which are located at the corneal-conjunctival boundary, or limbus. More specifically, this model predicts that the generation of new corneal epithelial cells at the limbus physically holds back the surrounding conjunctival epithelial cells.
Nevertheless, it remains possible that molecular sign-posts may also play a crucial role. If such sign-posts exist, then this behaviour might be retained by cells when examined in vitro. Two cell lines will be chosen for this study owing to their availability and reproducible characteristics compared with primary cell lines. If time permits however these studies will be repeated using a limited number of primary cell lines established via collaboration with colleagues at the Queensland Eye Institute.
Outcomes
These experiments are designed to answer one simple question: what is the respective behaviour of corneal and conjunctival epithelial cells when grown together in vitro? Surprisingly, a study of this kind appears to have never been attempted and as such the outcomes should therefore be worthy of publication, even if negative results are obtained. Assuming however that the results are positive, this will pave the way for a fresh approach to studying the corneal-conjunctival interface. In particular, it is anticipated that these experiments will produce valuable data for subsequent mathematical modelling of corneal-conjunctival cell interactions and thus form the basis for future grant applications with colleagues from the School of Mathematical Sciences including Professor Graeme Pettet.
2. To study the expression of Eph and ephrin family proteins across the cornea-conjunctiva boundary in situ.
Our hypothesis is that a differential expression pattern will be observed, consistent with a potential role in promoting formation of a barrier between the conjunctiva and cornea. Based upon recent studies in mouse tissues 2, 3, we anticipate that the human corneal epithelial cells will express ephrin-B1. It remains to be seen if appropriate Eph receptors to support partitioning of the two tissues are expressed by the conjunctiva.
Rationale
The central hypothesis for this study is based upon over ten years of Eph/ephrin research in numerous vertebrate tissues4-6. Eph proteins belong to the largest family of receptor tyrosine kinases and interact with ligands known as ephrins. Both proteins are normally expressed within the cell surface and receptor-ligand binding results in bi-directional signalling in adjacent cell types. Within the developing embryo Eph-ephrin interactions are responsible for guiding the movement of migrating cells and for establishing tissue boundaries, but an important role is now also envisaged in disease processes. One of the best known examples of this behaviour is seen within the developing nervous system of the chick embryo where migrating neural crest cells expressing EphB3 avoid contact with somitic tissue expressing ephrin-B14. Intriguingly, a limited number of studies have demonstrated expression of Eph/ephrin family members within the adult corneal epithelium of mice and humans (refer to Significance and Innovation), but the functional significance of this discovery has yet to be discerned.
Outcomes
Upon completion of these studies, we will have established data on the distribution of several key Eph/ephrin molecules across the corneal-conjunctival boundary. Such data will be highly novel with respect to studies of the human cornea and conjunctiva and as such should be suitable for publication in conjunction with data arising from the other Aims of this study. Of more immediate value, the molecules with a differential expression profile will be selected for study in Aim 3.
3. To investigate the respective in vitro responses of corneal epithelial and conjunctival epithelial cells to select Eph and ephrin proteins identified in Aim 2 as potential markers of tissue boundary.
Assuming that our above hypotheses prove correct, our third hypothesis will be that barrier-type responses can be recreated in vitro by allowing cultured cells to choose between surfaces coated with either Eph or ephrin proteins in accordance with the distinct patterns observed above by immunohistochemistry. For example, if ephrin-B1 is preferentially expressed in the cornea, then we would expect that conjunctival epithelial cells should avoid surfaces coated with this protein.
Rationale
The example of Eph/ephrin interactions in neural crest cells described above4 can be demonstrated in vitro, by showing that neural crest cells avoid contact with culture surfaces coated with ephrin-B1 protein. Thus, similar behaviour might be demonstrated in corneal/conjunctival cells if Eph/ephrin interactions are active in this tissue system.
Outcomes
Any demonstration of functional responses of corneal and/or conjunctival cells to Eph/ephrin molecules will be a highly novel and important discovery. In the short-term this data will be of a highly publishable nature, but the ultimate value of this research may well be to provide the foundation of a new family of therapeutics aimed at controlling common diseases of the ocular surface as discussed below.
Significance and innovation
The major clinical significance of this study resides in a condition known as pterygium, which affects approximately 1% of the Australian population7. Values as high as 13% have been measured for Australian males over the age of 60, and >30% for adults over 50 years in rural China (reviewed in8). A pterygium is a delta-shaped fibrovascular growth of conjunctival tissue which typically emerges from the nasal side of the peripheral cornea. While slow growing, the pterygia eventually extend onto the cornea and in doing so significantly impair vision. A range of medications aimed at reducing the growth of pterygia have been trialled, but treatment ultimately requires surgical excision.
Unfortunately, there is a high rate of recurrence in many centres, with secondary lesions often displaying more aggressive behaviour. Strong links have emerged between levels of UV light exposure and the occurrence of pterygium, but the underlying cellular basis remains unclear9.
To the best of our knowledge, no-one has previously suggested a direct role for Eph/ephrin type interactions in the development of pterygium, however a study by John-Aryankalayil et al (2006)8 detected a two-fold decrease in ephrin-A1 expression when comparing pterygial tissue with normal conjunctiva. Two more recent studies by Kojima et al (2007)2, 3 have demonstrated expression of ephrin-A1, ephrin-B1, Eph-A3, Eph-B1 and Eph-B4 in the normal corneal epithelium of mice, but did not report on the relative expression of these markers in the adjacent conjunctiva.
Thus, there is good evidence of Eph/ephrin expression within the normal and diseased cornea, but little data is available on human tissues or in comparison to surrounding tissues. Moreover, no studies have been made of the affects of Eph/ephrin molecules on the respective behaviour of corneal or conjunctival cells in vitro. The present study will therefore be novel with respect to: (1) studies in human tissue, (2) comparison of cornea with conjunctiva, and (3) functional studies.
References
- Harkin DG, Barnard Z, Gillies P, Ainscough SL, Apel AJ. Analysis of p63 and cytokeratin expression in a cultivated limbal autograft used in the treatment of limbal stem cell deficiency. Br J Ophthalmol 2004;88:1154-1158.
- Kojima T, Chang JH, Azar DT. Proangiogenic role of ephrinB1/EphB1 in basic fibroblast growth factor-induced corneal angiogenesis. The American journal of pathology 2007;170:764-773.
- Kojima T, Chung TY, Chang JH, Sayegh R, Casanova FH, Azar DT. Comparison of EphA receptor tyrosine kinases and ephrinA ligand expression to EphB-ephrinB in vascularized corneas. Cornea 2007;26:569-578.
- Krull CE, Lansford R, Gale NW, et al. Interactions of Eph-related receptors and ligands confer rostrocaudal pattern to trunk neural crest migration. Curr Biol 1997;7:571-580.
- Lackmann M, Boyd AW. Eph, a protein family coming of age: more confusion, insight, or complexity? Science signaling 2008;1:re2.
- Pasquale EB. Eph-ephrin bidirectional signaling in physiology and disease. Cell 2008;133:38-52.
- Wlodarczyk J, Whyte P, Cockrum P, Taylor H. Pterygium in Australia: a cost of illness study. Clinical & experimental ophthalmology 2001;29:370-375.
- John-Aryankalayil M, Dushku N, Jaworski CJ, et al. Microarray and protein analysis of human pterygium. Molecular vision 2006;12:55-64.
- Hirst LW. The treatment of pterygium. Survey of ophthalmology 2003;48:145-180.
- Araki-Sasaki K, Ohashi Y, Sasabe T, et al. An SV40-immortalized human corneal epithelial cell line and its characterization. Investigative ophthalmology & visual science 1995;36:614-621.
- Diebold Y, Calonge M, Enriquez de Salamanca A, et al. Characterization of a spontaneously immortalized cell line (IOBA-NHC) from normal human conjunctiva. Investigative ophthalmology & visual science 2003;44:4263-4274.
- Study level
- PhD
- Supervisors
- QUT
- Organisational unit
Science and Engineering Faculty
- Research area
- Contact
- Please contact the supervisor.
Dr Neil Richardson
Associate Professor Damien Harkin
